To gain a better understanding of the process of DNA replication in eukaryotic cells, I propose a study of the replication apparatus of the yeast Saccharomyces cerevisiae. The object of the study will be small circular DNA plasmids which replicate in yeast under nuclear control. Both an endogenous yeast plasmid and recombinant DNA plasmids containing functional yeast replicons cloned in Escherichia coli vectors will be used. Assays will be developed for the proteins involved in the replication process, the proteins will be purified using established techniques, and the mechanisms of action of the purified proteins will be investigated. The purified proteins will be assayed for activities predicated to be involved in the replication process inlcuding DNA binding, DNA-dependent nucleoside triphosphatases, DNA polymerase, RNA polymerase, RNAse, DNAse, DNA ligase, and DNA untwisting activities, in an attempt to determine the role of each protein in the overall mechanisms. Mutant plasmids will be constructed and the effects of the mutations on the replication of the plasmids in yeast and in the test tube will be evaluated in comparison with the unmodified templates. I will also investigate the influence of yeast mutations involved with host cell DNA synthesis on the duplication of the plasmids containing yeast chromosomal replicons. Replication extracts from the host mutant strains will be prepared and a correlation of the mutations with specific replication proteins will be attempted. The influence of drugs and hormones known to affect DNA replication will be measured using the cell-free replication system.